Spreading and migration of VSOP/Hv1^−/− neutrophils. Bone marrow neutrophils were seeded on BSA-coated Greiner 96-well plates and fMIVIL was added to promote chemokinesis. (A) Representative phase-contrast images of neutrophils before (top) and after (bottom) stimulation with fMIVIL+PMA. The combination of fMIVIL and PMA induced 100% of the cells to spread. (B) Percentage of spread cells among naive WT and VSOP/Hv1^−/− neutrophils. Data are means ± SD of 107 WT and 94 VSOP/Hv1^−/− neutrophils from three independent experiments. **, P < 0.01, unpaired Student's t test. (C) Migration tracings of nine WT and nine VSOP/Hv1^−/− neutrophils exposed to 10 µM fMIVIL for 45 min. (D) Mean migration speed (in micrometers per minute) of neutrophils exposed to DMSO, to fMIVIL alone, to fMIVIL together with PMA, or to fMIVIL together with 100 nM of the Ca²⁺ ionophore ionomycin in a buffer containing 5 µM Ca²⁺. Note that PMA prevented both WT and VSOP/Hv1^−/− neutrophils from migrating effectively upon fMIVIL stimulation, whereas ionomycin restored normal migration in VSOP/Hv1^−/− neutrophils. Data are mean ± SEM of 13–33 individual tracings for each condition from three to five independent experiments. ***, P < 0.0001; **, P < 0.001; *, P < 0.05, unpaired Student's t test. Five WT and five VSOP/Hv1^−/− mice were sacrificed for this experiment.